The goal of this grant proposal is to improve diagnostic testing for Lyme disease and to identify spirochetal and host factors that lead to more severe disease. This goal will be carried out by the analysis of samples from large retrospective and prospective series of well-characterized patients with Lyme disease. For this project, a leading candidate second-generation serologic test, an IgM OspC (C10)-peptide ELISA and an IgG VIsE (IR6)-peptide ELISA, will be assessed prospectively' and compared with the results obtained by whole-cell sonicate ELIoA and Western blot in patients with early or late Lyme disease and in control subjects. Antibody responses to novel glycolipid antigens of B. burgdorferi will be explored to learn whether serial determinations of these responses after antibiotic treatment may serve as a potential surrogate marker for spirochetal killing. The value of quantitative PCR as a diagnostic test will be determined for patients with Lyme disease as compared with standard PCR and culture. The spirochetal burden in skin biopsy samples of erythema migrans (EM) skin lesions and spirochetal virulence factors in isolates from these lesions will be delineated, and the results will be correlated with clinical markers of spirochetal dissemination and severity of infection. Finally, cytokine and chemokine profiles in EM lesions from these patients will be determined and correlated with clinical markers of spirochetal dissemination and severity of infection and with blood test results of cellular and humoral immune function. These studies are likely to lead to more accurate tests for the serodiagnosis of Lyme disease, for direct detection of the spirochete, and for the assessment of spirochetal killing. In addition, greater knowledge of spirochetal virulence and host factors associated with severe disease may allow the development of laboratory markers for patients who would benefit from more intensive treatment.